Hancock Laboratory Methods,Department of Microbiology and Immunology,
University of British Columbia, British Columbia, Canada
http://www.cmdr.ubc.ca/bobh/showmethod.php?methodid=49
Zwittergent 3-14 was obtained from Calbiochem. The CMC of this detergent is 0.012% (w/v), molecular weight = 363.6. All solubilization steps used 0.1% Zwit 3-14 unless otherwise indicated. Total protein concentration in any solubilization step should not exceed 10 mg/ml for maximum efficiency of solubilization. Protocol #1 will not produce as clean a preparation of proteins as will protocol #2 which involves extensive solubilizations of the outer membrane proteins.
PROTOCOL 1:
- Prepare envelopes from whole cells by French pressing. Separate whole cells from broken ones by low speed centrifugation (5,000 x g for 15 minutes). Pellet envelopes by high speed centrifugation (150,00 x g for 1 hour).
- Resuspend envelope pellet in an adequate volume of 0.5 mg/ml lysozyme in 20 mM MOPS pH=7.5 by sonication 3 x 10 second bursts.
- Add Zwit 3-14 to a final concentration of 1% (w/v) and homogenize by sonication.
- Centrifuge 150,000 x g for 1 hour.
- Go to step 10 of protocol #2.
PROTOCOL 2:
- Prepare envelopes as above or prepare outer membranes as per usual protocol.
- Resuspend envelope pellet in an adequate volume of 0.1% Zwit 3-14 in 20 mM MOPS buffer, pH=7.5 by sonicating 3 x 10 second bursts.
- Centrifuge 150,000 x g for 1 hour. (41k in 70.1 Ti or 40k in 60 Ti)
- Retain supernatant and resuspend pellet in an adequate amount of 0.1% Zwit 3-14, 50 mM EDTA in 20 mM MOPS pH=7.5 by sonication.
- Centrifuge 150,000 x g for 1 hour.
- Retain supernatant and resuspend pellet in an adequate amount of 0.1% Zwit 3-14, 0.4 M NaCl in 20 mM MOPS pH=7.5 by sonication.
- Centrifuge 150,000 x g for 1 hour.
- Retain supernatant and resuspend pellet in an adequate amount of 1.0% Zwit 3-14 in 20 mM MOPS pH=7.5 by sonication.
- Check all fractions for the protein you seek by SDS-PAGE. OprP is primarily solubilized in Zwit 3-14 with EDTA and OprF is solubilized in Zwit 3-14 with NaCl.
- Load fractions containing protein of interest on Q-Sepharose column. This column is an anion exchange resin similar to the Mono-Q FPLC (Pharmacia) resin and can be used to directly scale up from the Mono-Q column. A bed volume of approximately 130 ml was used for purification of large amounts of protein (ie. preps from 100 l of cells), however, this size of column requires a large gradient volume of 5.2 l. On the large column sample loadings of between 15 and 25 mls were used (sample concentration approximately 10 mg/ml). A gradient elution with buffers: Buffer A = 0.1% Zwit 3-14, 20 mM MOPS pH=7.5, 10% methanol; Buffer B = same as A with 1.0 M NaCl. Run at a flow rate of 6 ml/min. The gradient profile was linear from 0 to 45% buffer B for the first 30% of the run followed by a plateau of 45% B for an additional 30% of the run, then increasing linearly (in approximately 10% of total run) to 100% B to elute off any remaining sample. The porin proteins tend to elute in the last major peak before increasing to 100% B.
- Fractions containing the protein of interest are pooled and washed with buffer A in Centricon units (Millipore) to wash away the salt and concentrate the sample.
- Sample is then applied to Mono-Q column (Pharmacia) and eluted with a linear gradient using the same buffers as above.
- Fractions of interest are again washed and concentrated with the Centricon units and either lyophilized or stored at -70 C to prevent degradation of the proteins.
SOLUBILIZATION OF OUTER MEMBRANE D2 PROTEIN:As per
Protocol #2 but the pellets are solubilized in this series of solutions:
Step 2: 1. 1% Zwit. 3-14 in 20mM MOPS pH 7.5
Step 4: 2. 1% Zwit. 3-14 in 20mM MOPS pH 7.5
Step 6: 3. 1% Zwitt 3-14: in 20mM MOPS pH 7.5, 10mM EDTA, 0.1 m NaCl:
Step 8: 4. dH
2O
上一篇：Protease Digestion of Outer Membranes蛋白酶水解细胞外膜 下一篇：Penicillan Binding Protein Assay青霉素与细胞膜蛋白结合实验
